Leurosine



United States Patent 3,370,057 LEUROSINE I Gordon H. Svohoda,Indianapolis, Ind., assiguor to Eli This is a continuation-in-part of mycopending US. patent application Ser. No. 43,499, filed July 18, 1960(which was a continuation-in-part of my US. application Ser. No.756,761, filed Aug. 25, 1958, and of my copending application Ser. No.129,567, filed Aug. 7, 1961 all three now abandoned.

The invention described and claimed herein relates to a novel substance,and to methods for its preparation and purification.

The novel substance provided by this invention is denominated leurosine,and is obtained from certain plants belonging to the family Apocynaceae.It is a white, crystalline, weakly-basic, nitrogen-containing compoundwhich decomposes on heating at about 200-205 C.

Leurosine is soluble in most of the common oxygenated organic solvents,such as acetone, alcohol, ethyl acetate, etc., as well as in aromatichydrocarbon solvents, such as benzene and toluene, and in chlorinatedaliphatic solvents such -as chloroform and ethylene dichloride.Leurosine is, however, insoluble in aliphatic hydrocarbon solvents onthe one hand and water on the other. Leurosine is a weakly basicsubstance and hence is soluble in dilute acid solution, but isrelatively unstable in such solutions, especially in mineral acidsolutions having a low pH.

.Leurosine crystallizes from water-containing solvents in the form of ahydrate containing eight molecules of water. The hydrate is somewhatunstable and loses its water of hydration on standing. The anhydrouscrystalline form of leurosine, however, which can be obtained from thehydrate by drying at an elevated temperature in vacuo appears to berelatively stable.

As with other weak bases which are alkaloidal in nature, leurosine givesa creamy white precipitate with Mayers reagent, an orange color withDragendorfis reagent, a yellow color with the Van Urk reagent, and apink color with ceric ammonium sulfate dissolved in syrupy phosphoricacid.

IDENTIFICATION OF LEUROSINE Percent Carbon 68.11 Hydrogen 7.30 Nitrogena 7.10 Oxygen 17.34

(3) The molecular weight of leurosine octahydrate, as determined byX-ray data, is about 955, giving a weight for leurosine of about 809.These data establish the following empirical formula for leurosine: C HO N (4) Analyses for various groups in leurosine were as follows:

Percent C-methyl 4.91 Methoxyl 15.31 N-methyl 1.43

These values, when taken in connection with the above molecular weight,indicate that each molecule of leurosine contains one C-methyl group,four methoxyl groups, and one N-methyl group.

(5) The ultraviolet absorption spectrum of an ethanol solution ofleurosine octahydrate shows maxima at 214 and 259 In, with shoulders at288 and 296 m and a minimum at 246 [fl 1., the maxima having molecularextinction coelficients of 575 and 175, respectively.

(6) Leurosine octahydrate has the following specific rotation inchloroform solution:

(7) The infrared absorption spectrum, over the range from 2 to 15microns of a chloroform solution of leurosine octahydrate, is presentedin the accompanying drawing. The infrared absorption spectrum exhibitscharacteristic absorption maxima at the following wave lengths(expressed in microns): 2.90, 3.35, 3.40, 3.50, 5.74, 6.18, 6.65, 6.85,6.97, 7.28, 7.50, 7.73, 8.1, 8.64, 8.72, 8.83, 8.92, 9.02, 9.15, 9.40,9.59, 10.21, 10.41, 10.56, 10.75, 10.88, 11.10, 11.28, and 12.17.

Two additional absorption maxirna can be detected in the infrared regionof the spectrum at 8.06 and 8.7 microns when carbon disulfide isemployed as the solvent. The peaks in this region are obscured whenchloroform is used as the solvent because of the strong absorption ofchloroform in this region of the spectrum.

(8) Leurosine can be further characterized by paper chromatographicmethods. For such characterization, a paper treated with phosphatebuffer is used, and the developing solvent is either isobutanolsaturated with water or n-amyl alcohol saturated with water. The treatedpaper is prepared as follows: A suitable paper (Whatman No. 1 paper) isdipped in a buffer prepared from g. of fresh potassium hydroxide pelletsdissolved in about 800 ml. of water to which are added 60 ml. ofconcentrated phosphoric acid in 100 ml. of water. The pH of the bufferis adjusted to 3.0 by the addition of phosphoric acid, about 10 ml.usually being sufiicient, and water is added to make one liter of buffersolution. The paper is dipped oped for about 24 hours at about 74 F. Thepresence of leurosine is detected either by scanning the paper with lowwave length ultraviolet light or by applying Dragendorfis reagent to thepaper. Dragendorffs reagent is prepared according to the procedure ofThies and Reuther, Naturwissenschaften, vol. 41, page 230 (1954), andvol. 42, pages 462 and 487 (1955). In short wave length ultravioletlight, leurosine appears as a dark spot with very slight fluorescence;and with Dragendorifs reagent, leurosine appears as a red-orange spot ona yellowish-orange background. The R, value of leurosine, .as determinedby means of the above procedure, is 0.51 in an isobutanol-water solventsystem using the phosphatebuffered paper, and 0.28 in an n-arnylalcohol-water solvent system.

(9) Thin layer chromatography can also be used to identify leurosine andthus to signify its presence or absence in various plant extracts. Incarrying out this chromatographic procedure a thin layer of fluorescent(activated with zinc sulfide) silica gel is slurried with 0.5 N lithiumhydroxide, deposited as a thin layer on glass, and the resulting glasswith its layer of silica is dried. A solution containing 12 mg. per ml.of leurosine is prepared in chloroform and 20A of this solution isspotted on the thin layer at the origin, and the thin layer is thenplaced in a standard jar. The developing solvent contains parts ofdiethylamine, 100 parts of chloroform,, and 200 parts of benzene. Thedevelopment of the chromatogram can be followed by short wave lengthultraviolet light. After the chromatogram has been developedsufficiently, the plate containing the thin layer is removed from thechamber and dried. The spot corresponding to leurosine can be detectedby use either of ultraviolet light or of the abovementioned reagentcomprising ceric ammonium sulfate in syrupy phosphoric acid.

In general, vincaleukoblastine is the only alkaloid which runs withleurosine through most chromatographic systems. The above procedure,however, is quite satisfactory for the separation of these twoalkaloids.

(10) Leurosine octahydrate crystallizes in the monoclinic system withunit cell dimensions as follows:

(11) The specific gravity of crystalline leurosine octahydrate is 1.262.

(12) A powder X-ray diffraction pattern using unfiltered chromiumradiation and a wave length value of 2.2896 A. in calculating theinterplanar spacing, gives the following values:

TABLE I.X-RAY POWDER DIFFRAOTION DATA Interplanar Relative InterplanarDistance Distance in A. Intensity in A. Calculated From Unit CellDimensions 15. 8 50 15. 78 13. 4 40 13. 21 11.0 50 10. 82 9. 81 9. 56 9.29 20 9. 28 8. 77 .05 8. 76, 8. 81 8.10 1.00 8.11, 8.00 7. 18 50 7. 186. 59 75 6. 61 6.34 2O 6. 38 6. O9 20 6. 01 5. 47 10 5. 56 5. 5. 26, 5.29 4. 94 05 4. 94 4. 73 10 4. 77, 4. 77, 4. 78, 4. 70 4. (r1 30 4. 64,4. (59 4.13 05 4.12, 4. 16, 4.11 4. 03 05 4. 06 3. 93 10 3. 95, 3. 903.76 .10 3.78, 3.78 3. 66 .05 3. G6, 3. 66, 3. 63 3. 51 05 3. 52 3. 38.05 3. 41, 3. 3. 33 .05 3. 35, 3. 37 3. 17 .05 319,319

PREPARATION OF LEUROSINE The plant sources from which leurosine can beobtained are those belonging to the family Apocynaceae, and particularlythe genus Vinca. Especially productive sources include Cathamnthusroseus, also popularly known as Vinca rosea or Lochnera rosea andCatharamhus Icmceus or Vinca lancea. The recovery of leurosine therefromis conveniently carried out by the following procedure:

The plant source, in the form of either the whole plant or its leaves,is dried and ground to a powder, which is then wetted with a solution ofan organic acid in water, said solution having a pH in the range 2 to 3.The wetted crude drug is extracted with benzene, and the benzene extractis separated and concentrated by evaporation in vacuo to a workablevolume. An aqueous solution of an organic acid is then added to thisconcentrate and the remaining benzene is removed therefrom by steamdistillation under reduced pressure, yielding an acidic solution ofalkaloidal material having in suspension essentially non-alkaloidalmaterial. The suspended solids are separated by filtration. The acidiclayer is next extracted with ethylene dichloride to remove inactivealkaloidal int purities. The acidity of the aqueous phase is thenadjusted to a pH in the range 7.5 to 8.5, preferably using am moniumhydroxide. Contacting the now alkaline layer with ethylene dichlorideserves to extract the active alka loids into the organic layer. Theethylene dichloride layer is separated and dried and the ethylenedichloride removed by evaporation in vacuo. The resulting residue isdried and is then dissolved in benzene, and chromatographed over analumina column to separate the leurosine from companion alkaloids.Purified leurosine is eluted from the column with a benzene-chloroformsolvent mixture, after less polar alkaloids have been eluted withbenzene or ether or other relatively non-polar solvents.

Leurosine, as such, does not appear to exist in the plant or in thecrude extracts of the prior art, since it has not been possible todemonstrate its existence in such extracts by means of paper stripchromatography or thin layer chromatography using very sensitive colorreagents.-

Leurosine is useful in the study of the origin and mech-- anism ofcancer, since it inhibits the growth of various transplanted tumors inmice, particularly transplanted leukemias. Table II, which follows, setsforth the tumor spectrum of leurosine in mice against varioustransplanted leukemias and ascites tumors.

TABLE IL-COMPOSITE RESULTS OF TUMOR SPECTRA STUDIES WITH LEUROSINEStrain Dosage in N o. of Doses Prolongation,

rug/kg. Percent Leukemias 30. 00 10 20 9. 38 1O 68 30. 00 3 30.00 10 213. 75 10 20 12. 60 3 33 30.00 10 36 30. 00 10 56 9. 28 10 0 30. 00 10 359. 38 10 0 Ascites Tumors 1 3 survivors.

Leurosine also inhibits the growth of two transplanted solid tumors;viz., Sarcoma in mice, where a dose of 10.5 mg./kg. on each of 10successive days gives 35% inhibition of the tumor; and Walker carcinoma265 in rats, where a dose of 15 mg./kg. on each of 10 successive daysgives a 49% inhibition of the tumor.

As can be seen from Table II above, leurosine is quite effective inprolonging the life of experimental animals in which leukemias andascites tumors have been transplanted. Its spectrum of activity issubstantially different from that found with either vincaleukoblastine(vinblastine), leurocristine (vincristine), or leurosidi-ne(vinrosidine), as Well as with derivatives of these oncolytic alkaloids.This difference in oncolytic activity among the various Vinca alkaloidsreflects a difference in chemical structure. Thus, leurosine, in companywith other Vinca alkaloids which are perhaps not sufficiently activeever to meet the criteria established for marketing :as a remedy againstmalignancies in humans, is nevertheless extremely useful in pointing outto those skilled in the art the minimal structural requirements for theinhibition of transplanted tumors among these substances. Thus,knowledge of the structure of leurosine and of its anti-tumor activitycan aid materially in the preparation of more. active compounds on theone hand, and in fur hfi ing the knowledge of the mitotic process inmalignant cells on the other.

Example I 1500 g. of dried ground plant of Vinca rosea were intimatelymixed with 1000 ml. of a 2 percent tartaric acid solution, and themixture was extracted with three 9-liter portions of benzene. Thebenzene extracts were combined and were concentrated in vacuo to about1500 ml. The concentrate was mixed with one liter of 2 percent tartaricacid and the mixture was steam-distilled under reduced pressure untilall of the benzene had distilled over. The precipitate produced therebywas separated from the equeous phase and dissolved in hot methanol, asecond l-liter portion of 2 percent tartaric acid solution was added,and the mixture was steamdistilled under reduced pressure until all themethanol had distilled. The two tartaric acid solutions from the steamdistillations were combined and washed with three l-liter portions ofethylene dichloride, which were discarded. The washed aqueous phase wasbrought to a pH of about 8.5-9.5 by the addition of 28 percent aqueousammonium hydroxide, and was extracted with three l-liter portions ofethylene dichloride. The ethylene dichloride extracts were combined,were dried, and were evaporated in vacuo, yielding a residue of 3.35 g.of a light brown powder.

1.5 g. of the residue Were dissolved in ml. of henzene, and the solutionwas passed over a chromatographic adsorption column containing 50 g. ofalumina (Alcoa activated alumina, Grade F- obtainable from The AluminumCompany of America, Pittsburgh, Pa.) which had previously been shakenfor about 20 minutes with a mixture of 100 ml. of benzene containing 1.5ml. of 10 percent acetic acid.

The column was developed by washing it with 2100 ml. of benzene. Thecolumn was then washed sequentially with 300 ml. of benzene-chloroformsolvent (95:5 by volume) and 800 ml. of benzene-chloroform solvent(75:25) to remove indeterminate impurities. The leurosine was elutedfrom the alumina by passing over the column 900 ml. ofbenzene-chloroform solvent (50:50). The eluate was evaporated to drynessin vacuo, leaving an amorphous residue of 113 mg. of leurosine. Theresidue was treated with a few ml. of methanol in which it quicklydissolved, but from which it quickly precipitated in crystalline form.Because of the afiinity of leurosine for water, and the presence oftraces of water in the solvents, the leurosine was obtained in the formof its octahydrate. Although the material as obtained was substantiallypure, it was further purified by recrystallizing it from hot methanolsolution. The hydrated leurosine obtained decomposed at about 200205 C.

Anhydrous leurosine was obtained by heating the hydrated material in anAbderhalden drier at 130 C. over phosphorus pentoxide for about 3 hours.The anhydrous leurosine decomposed at about 200-205" C.

Example II 9 kg. of ground, dried, whole Vinca Rosea plants weredefatted by stirring with two 45-liter portions of hexane and thendiscarding the hexane extracts. The defatted solids were moistened with6 liters of aqueous 2 percent tartaric acid solution and were extractedby stirring with three successive 48-liter portions of benzene. Thebenzene extracts were combined and were concentrated in vacuo to avolume of about 9 liters. To the concentrate were 6 added 12 liters ofaqueous 2 percent tartaric acid solution, and the organic solvent wasremoved by steam distillation under reduced pressure, during whichoperation the leurosine and vincaleukoblastine dissolved in the aqueousacid layer in the form of their tartrate salts. Acid-insoluble materialwas separated by filtration. The filter cake was dissolved in methanol,12 liters of aqueous 2 percent tartaric acid solution were added, andthe above steam distillation procedure was repeated. The tartaric acidlayer was filtered to remove undesirable insoluble material and thefiltrate was combined with the previous tartartic acid filtrate. Thecombined filtrates were extracted with two 6-liter portions of ethylenedichloride. Tetrahydroalstonine, vindoline, and related alkaloids wereextracted into the ethylene dichloride and were thus separated from theleurosine and vincaleukoblastine. The acidic aqueous solution was thenmade alkaline to litmus with ammonium hydroxide. Extraction of theammoniacal solution with 6 liters of ethylene dichloride removed theweakly basic alkaloid fraction containing leurosine andvincaleukoblastine. Evaporation of the ethylene dichloride extract todryness yielded about 20 g. of amorphous alkaloids. 10 g. of thisresidue were dissolved in benzene and the benzene solution was subjectedto a preliminary chromatographic separation using as the solid phase 400g. of alumina (Alcoa alumina, grade A20) which had previously beendeactivated by treatment with 12.5 ml. of 10 percent acetic acid.Elution was carried out with a series of solvents, as set forth below inTable III, the volume of each chromatographic fraction being arbitrarilyset at 500 ml. Table III sets forth the results of the chromatographicseparation procedure. In the table, column 1 gives the number of thefraction, column 2 the eluting solvent, and column 3 the major alkaloidobtained from that fraction.

TABLE III Fraction Eluting Solvent Alkaloid Catharanthine. Vindolinine.Ajmalicine. Vindoline. eloroform .1) Leurosine.

Vinealeukoblastine.

1 crystallized as the dihydroehloride salt. 2 crystallized as thesullate salt.

I claim:

1. Leurosine, a crystalline, weakly basic, nitrogen-containing compoundhaving a molecular weight of 809 and showing upon analysis the presenceof 68.11 percent carbon, 7.30 percent hydrogen, 7.10 percent nitrogen,and 17.34 percent oxygen, the said values establishing the empiricalformula C H O N having an N-methyl group content of 1.43 percent, aC-methyl group content of 4.91 percent, and a methoxyl group content of15.31 percent; showing pK,, values in water of 5.5 and 7.5; itsoctahydrate having ultraviolet absorption maxima at 214 and 259 mg withshoulders at 288 and 296 mm, and a minimum at 246 m the maxima havingmolecular extinction coefiicients of 575 and 175, respectively; itsoctahydrate exhibiting in a chloroform solution in the infrared regionover the range of about 2 to about 15 microns, characteristic peaks at2.90, 3.35, 3.40, 3.50, 5.74, 6.18, 6.65, 6.85, 6.97, 7.28, 7.50, 7.73,8.1, 8.64, 8.72, 8.83, 8.92, 9.02, 9.15, 9.40, 9.59, 10.21, 10.41,10.56, 10.75, 10.88, 11.10, 11.28 and 12.17 microns; its octahydratehaving the following specific rotation in chloroform solution: [a] =+72(c.=1); having a decomposition point in the range 200205 C.; itsoctahydrate having a specific gravity of 1.262; and its octahydratecrystallizing in the monoclinic system with the following unit celldimensions: a =26.64, b =9.28, 0 :15.90, B=9710'.

(References on following page) 7 8 References Cited Svoboda, Jour. Amer.Pharmaceut. Assn, v01. 47 A 1958) p. 834. i. t hhnson et al CancerRfisearch V01 (Angus Hertz Proc. Soc. Ex. Biol. and Med., Vol. 105(1960), 1960), pp. 1016-22. p 281 Hodes et a1.: Cancer ChemotherapyReports (April 1963), p. 53. 5 JAMES A. PATTEN, Primary Examiner.

Beer British Empire Cancer Campaign, 33rd Annual WALTER MODANCE JOHN D.R ANDOLPH, Rept. (1955), p. 487. Examiners.

1. LEUROSINE, A CRYSTALLINE, WEAKLY BASIC, NITROGEN-CONTAINING COMPOUNDHAVING A MOLECULAR WEIGHT OF 809 AND SHOWING UPON ANALYSIS THE PRESENCEOF 68.11 PERCENT CARBON, 7.30 PERCENT HYDROGEN, 7.10 PERCENT NITROGEN,AND 17.34 PERCENT OXYGEN, THE SAID VALUES ESTABLISHING THE EMPIRICALFORMULA C46H58O9N4; HAVING AN N-METHYL GROUP CONTENT OF 1.43 PERCENT, AC-METHYL GROUP CONTENT OF 4.91 PERCENT, AND A METHOXYL GROUP CONTENT OF15.31 PERCENT; SHOWING PKA'' VALUES IN WATER OF 5.5 AND 7.5; ITSOCTAHYDRATE HAVING ULTRAVIOLET ABSORPTION MAXIMA AT 214 AND 259 MU WITHSHOULDERS AT 288 AND 296 MU, AND A MINIMUM AT 246MU, THE MAXIMA HAVINGMOLECULAR EXTINCTION COEFFICIENTS OF 575 AND 175, RESPECTIVELY; ITSOCTAHYDRATE EXHIBITING IN A CHLOROFORM SOLUTION IN THE INFRARED REGIONOVER THE RANGE OF ABOUT 2 TO ABOUT 15 MICRONS, CHARACTERISTIC PEAKS AT2.90, 3.35, 3.40, 3.50, 5.74, 6.18, 6.65, 6.85, 6.97, 7.28, 7.50, 7.73,8.1, 8.64, 8.72, 8.83, 8.92, 9.02, 9.15, 9.40, 9.59, 10.21, 10.41,10.56, 10.75, 10.88, 11.10, 11.28 AND 12.17 MICRONS; ITS OCTAHYDRATEHAVING THE FOLLOWING SPECIFIC ROTATION IN CHLOROFORM SOLUTION:(A)D36*=+72* (C.=1); HAVING A DECOMPOSITION POINT IN THE RANGE200-205*C; ITS OCTAHYDRATE HAVING A SPECIFIC GRAVITY OF 1,262; AND ITSOCTAHYDRATE CRYSTALLIZING IN THE MONOCLINIC SYSTEM WITH THE FOLLOWINGUNIT CELL DIMENSIONS: AO=26.64, BO=26.64, BO=9.28, CO=15.90, B=97*10''.